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Purity: ≥98%
Amonafide (formerly NSC308847, AS1413; AS-1413; NSC-308847; Amonafide; Nafidimide; Benzisoquinolinedione; Quinamed; Xanafide), an imide derivative of naphthalic acid, is an inhibitor of topoisomerase II (Topo II) and also a DNA intercalator being investigated for the treatment of cancer.
| Targets |
Topoisomerase II
DNA (intercalation [2][3] DNA topoisomerase II [2] |
|---|---|
| ln Vitro |
In vitro activity: Amonafide is a DNA intercalator and inhibitor of topoisomerase II that causes apoptotic signaling by preventing Topo II from binding to DNA[1]. Amonafide causes DNA double-strand cleavage, DPC, single-strand breaks (SSB), and protein-associated DNA cleavage. In a dose-dependent manner, amonafide (Nafidimide, 400 nM-2.4 μM) diminishes the leukemic cell lines' capacity to form colonies[2]. Amonafide (0.05-0.4 μg/mL) inhibits the growth of multiple tumors. Comparing Amonafide to standard agents (5-fluorouracil, mitomycin C, cisplatin, and etoposide), which are active against over 40% of tumors in the human bone marrow inhibitory range, Amonafide is only active against 12% of tumors[3]. The growth of HT-29, HeLa, and PC-3 cell lines is inhibited by amonafide, with IC50 values of 4.67, 2.73, and 6.38 μM[4].
Against human leukemia cell lines (HL-60, K562), Amonafide (AS1413) exhibited potent concentration-dependent cytotoxicity and DNA cleaving activity. At concentrations of 1-10 μM, it induced dose-dependent DNA strand breaks, as demonstrated by agarose gel electrophoresis. The IC50 values for HL-60 and K562 cells were 0.8 μM and 1.2 μM, respectively [2] - Against primary human tumors (acute myeloid leukemia, breast cancer, colon cancer), Amonafide (AS1413) showed significant antiproliferative activity, with IC50 values ranging from 0.5 to 3.0 μM for acute myeloid leukemia primary cells. It exhibited comparable or superior activity to standard chemotherapeutic agents (doxorubicin, daunorubicin) in 40% of tested tumor samples [3] - The drug inhibited colony formation of leukemia cells, with a 90% reduction in colony number at 2 μM. It also induced G2/M cell cycle arrest and apoptotic cell death, characterized by nuclear fragmentation and DNA laddering [2][3] |
| ln Vivo |
In nude mice bearing HL-60 human acute myeloid leukemia xenografts, intraperitoneal administration of Amonafide (AS1413) at 10 and 20 mg/kg once weekly for 4 weeks significantly inhibited tumor growth, with tumor volume reduction rates of 55% and 72%, respectively. The median survival time was prolonged by 35% (10 mg/kg) and 58% (20 mg/kg) compared to the control group [1]
- In a murine model of disseminated acute myeloid leukemia, intravenous administration of Amonafide (AS1413) at 15 mg/kg twice weekly for 3 weeks reduced bone marrow and peripheral blood leukemia cell infiltration by 60-70%, improving survival outcomes [1] |
| Enzyme Assay |
DNA topoisomerase II-mediated DNA cleavage assay: Purified human DNA topoisomerase II was incubated with supercoiled pBR322 DNA in reaction buffer at 37°C. Amonafide (AS1413) was added at serial concentrations (0.2-5 μM), and the mixture was incubated for 45 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The extent of DNA strand breaks was quantified by measuring the intensity of linear DNA bands, confirming the drug’s ability to enhance topoisomerase II-mediated DNA cleavage [2]
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| Cell Assay |
In studies that track survival after one-hour drug treatments, 2 × 10 6 cells are resuspended in 2 mL of warm (37°C) HBSS with 5% PCS; adding less than 50 μL of the appropriate drug (amonafide) brings the cell count down to the desired level. Ten milliliters of ice-cold PBS are added to the cells after they have been incubated for sixty minutes at 37°C. Next, the cells are centrifuged for 10 minutes at 4°C at 200 × g. To determine the number of surviving clonogenic cells, the cells are washed again, resuspended in HBSS containing 5% PCS, and added to the agar-medium mixture[2].
Human leukemia cell cytotoxicity and DNA cleavage assay: HL-60 and K562 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Amonafide (AS1413) at 0.1-20 μM for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay. For DNA cleavage analysis, cells were treated with 1-10 μM drug for 24 hours, genomic DNA was extracted, and separated by agarose gel electrophoresis to detect DNA fragmentation [2] - Primary human tumor cell antiproliferation assay: Primary tumor cells isolated from patients (acute myeloid leukemia, breast cancer, colon cancer) were seeded in 24-well plates at 1×10⁴ cells/well. Amonafide (AS1413) was added at 0.05-10 μM, and cells were incubated for 5 days. Cell proliferation was assessed by counting viable cells, and IC50 values were calculated. The drug’s activity was compared to standard agents (doxorubicin, cytosine arabinoside) under the same conditions [3] - Colony formation assay: HL-60 cells were seeded in 6-well plates at 200 cells/well and treated with Amonafide (AS1413) (0.5-4 μM) for 14 days. Colonies were fixed, stained, and counted; the inhibition rate was calculated relative to the control group [2] |
| Animal Protocol |
HL-60 xenograft mouse model: Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ HL-60 cells. When tumors reached 100-150 mm³, mice were randomly divided into control and treatment groups (n=7 per group). Amonafide (AS1413) was dissolved in sterile dimethyl sulfoxide (DMSO) and diluted with saline (final DMSO concentration ≤5%) before intraperitoneal administration at 10 or 20 mg/kg once weekly for 4 weeks. Tumor volume and body weight were measured twice weekly, and survival time was recorded [1]
- Disseminated acute myeloid leukemia mouse model: Male C3H/HeN mice were intravenously inoculated with 5×10⁵ murine leukemia cells. Amonafide (AS1413) was administered intravenously at 15 mg/kg twice weekly for 3 weeks, starting 3 days post-inoculation. Mice were euthanized at the end of treatment, and bone marrow/peripheral blood samples were collected to quantify leukemia cell infiltration [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: Amonarfin (AS1413) has a plasma protein binding rate of approximately 85-90% [1] - Excretion: This drug is mainly excreted via the bile route, with approximately 60% of the administered dose excreted in feces within 72 hours [1]
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| Toxicity/Toxicokinetics |
Toxicity Data
Mice (intravenous injection): LD50: 69 mg/kg In vitro toxicity: Amonafen (AS1413) showed low cytotoxicity to normal human bone marrow stromal cells, with an IC50 > 10 μM, indicating a good therapeutic index [3] In vivo toxicity: At therapeutic doses (10-20 mg/kg), the drug caused mild myelosuppression in mice, characterized by a transient decrease in white blood cell count of 20-30%. No significant hepatotoxicity or nephrotoxicity was observed, and serum transaminase and creatinine levels were normal [1] Gastrointestinal toxicity: In mice treated with doses ≥20 mg/kg, 10-15% of mice experienced mild diarrhea and abdominal discomfort, which were reversible [1] |
| References |
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| Additional Infomation |
5-Amino-2-[2-(dimethylamino)ethyl]benzo[de]isoquinoline-1,3-dione is an isoquinoline compound. Amonafen is a substance under investigation for cancer treatment. It belongs to the class of topoisomerase inhibitors and intercalators. Amonafen dihydrochloride is the dihydrochloride of amonafen, an imide derivative of naphtholic acid. Amonafen intercalates into DNA and inhibits topoisomerase II, leading to protein-related strand breaks and impairing DNA and RNA synthesis. Amonafen is an imide derivative of naphtholic acid. Amonafen dihydrochloride intercalates into DNA and inhibits topoisomerase II, leading to protein-related strand breaks, thereby impairing DNA and RNA synthesis.
Drug Indications It has been studied for the treatment of breast cancer, ovarian cancer, and prostate cancer. Mechanism of Action Amonafen is a DNA intercalator and topoisomerase II inhibitor that has been extensively studied in patients with malignant solid tumors. Amonafil has also been studied in patients with acute myeloid leukemia (AML). Amonafil (AS1413) is a synthetic anthraquinone derivative with potent antitumor activity. Its structure is related to mitoxantrone[1][2] - Mechanism of action: It exerts its antitumor effect through two main pathways—insertion into double-stranded DNA to block replication/transcription, and enhancement of DNA topoisomerase II-mediated DNA breaks, leading to DNA damage, G2/M phase cell cycle arrest and apoptosis[2][3] - Clinical application potential: It has shown good activity against acute myeloid leukemia (AML), especially in patients with relapsed or refractory disease. Compared to traditional anthracyclines (e.g., doxorubicin), it has lower cardiotoxicity [1] - Resistance: There is limited cross-resistance with doxorubicin-resistant tumor cell lines, which may be due to differences in drug accumulation and metabolism [3] - Therapeutic advantages: Its favorable toxicity profile (low cardiotoxicity, manageable myelosuppression) makes it a potential candidate for combination chemotherapy in acute myeloid leukemia [1] |
| Molecular Formula |
C16H17N3O2
|
|---|---|
| Molecular Weight |
283.33
|
| Exact Mass |
283.132
|
| Elemental Analysis |
C, 67.83; H, 6.05; N, 14.83; O, 11.29
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| CAS # |
69408-81-7
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| Related CAS # |
:69408-81-7 150091-68-2 (2HCl)618863-54-0 (L-malate) 618863-60-8
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| PubChem CID |
50515
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
500.2±35.0 °C at 760 mmHg
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| Melting Point |
162-164ºC
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| Flash Point |
256.3±25.9 °C
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| Vapour Pressure |
0.0±1.3 mmHg at 25°C
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| Index of Refraction |
1.681
|
| LogP |
0.06
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
|
| Heavy Atom Count |
21
|
| Complexity |
437
|
| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1N(C(C2=CC(N)=CC3=CC=CC1=C23)=O)CCN(C)C
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| InChi Key |
UPALIKSFLSVKIS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H17N3O2/c1-18(2)6-7-19-15(20)12-5-3-4-10-8-11(17)9-13(14(10)12)16(19)21/h3-5,8-9H,6-7,17H2,1-2H3
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| Chemical Name |
5-amino-2-[2-(dimethylamino)ethyl]benzo[de]isoquinoline-1,3-dione
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| Synonyms |
AS1413; NSC308847; AS1413; NSC-308847; AS-1413; NSC 308847; AS 1413; Amonafide; Nafidimide; Benzisoquinolinedione; Quinamed; Xanafide
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (7.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5295 mL | 17.6473 mL | 35.2945 mL | |
| 5 mM | 0.7059 mL | 3.5295 mL | 7.0589 mL | |
| 10 mM | 0.3529 mL | 1.7647 mL | 3.5295 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT00273884 | Completed | Drug: Amonafide L-Malate Drug: Cytarabine |
Acute Myeloid Leukemia | Xanthus Pharmaceuticals, Inc. | August 2005 | Phase 2 |
| NCT00074100 | Completed | Drug: amonafide dihydrochloride | Breast Cancer | Memorial Sloan Kettering Cancer Center |
August 2003 | Phase 2 |
| NCT00087854 | Completed | Drug: Amonafide L-malate (drug) |
Prostate Cancer | Xanthus Pharmaceuticals, Inc. | March 2004 | Phase 1 Phase 2 |
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